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First published online 10 November 2004
doi: 10.1242/dev.01526
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Section of Integrative Biology, Section of Molecular, Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, 1 University Station C0930, Austin, TX 78712, USA
* Author for correspondence (e-mail: dparichy{at}mail.utexas.edu)
Accepted 11 October 2004
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Zebrafish, Pigment pattern, Evolution, Morphogenesis, Neural crest, Stem cell, Phylogeny
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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; Hall, 1999).
These cells arise along the dorsal neural tube during neurulation and then
disperse widely throughout the embryo
(Knecht and Bronner-Fraser,
2002; Halloran and Berndt,
2003). Among the cells and tissues derived from this transient,
migratory population are pigment cells, glia and neurons of the peripheral
nervous system, endocardial cushion cells, chromaffin cells of the adrenal
gland, smooth muscle, and bone and cartilage of the craniofacial skeleton
(Hörstadius, 1950;
Le Douarin, 1999). Not
surprisingly, in light of their many derivatives, neural crest cells are
associated with a wide range of inherited and acquired disorders ranging from
melanoma to neuroblastoma, Hirschsprung disease to Waardenburg syndrome, and
Treacher Collins syndrome to craniofacial dysmorphogenesis following fetal
ethanol exposure (Matthay,
1997; Amiel and Lyonnet,
2001; Ahlgren et al.,
2002; Chin, 2003;
Widlund and Fisher, 2003;
Farlie et al., 2004). Changes
in the patterning of neural crest cells and their derivatives are similarly
thought to underlie much of vertebrate diversity, from variation in pigment
pattern to variation in jaw morphology
(Kelsh, 2004;
Kulesa et al., 2004).
Given the biomedical and evolutionary significance of neural crest cells
and their derivatives, it is of paramount importance to identify the
mechanisms by which these cells are patterned to generate the particular forms
expressed by juveniles and adults. Most studies have focused on the early
patterning of neural crest cells during embryogenesis. Yet, recent studies
have demonstrated post-embryonic neural crest-derived stem cells in peripheral
nerves, gut and skin (Morrison et al.,
1999; Bixby et al.,
2002; Kruger et al.,
2002; Nishimura et al.,
2002; Iwashita et al.,
2003; Sieber-Blum and Grim,
2004; Sieber-Blum et al.,
2004; Joseph et al.,
2004). These findings suggest that the development and maintenance
of adult traits, as well as the evolution of these traits, may depend on
contributions from latent stem cells in addition to direct contributions from
neural crest cells at embryonic stages.
A useful system for studying the development and evolution of neural
crest-derived traits is the pigment pattern of teleost fishes
(Quigley and Parichy, 2002;
Parichy, 2003;
Kelsh, 2004). In the zebrafish
Danio rerio, an early larval pigment pattern develops during
embryogenesis as neural crest cells differentiate into early larval
melanophores and other pigment cell classes. This pattern is largely completed
by 3 days post-fertilization (dpf), and includes melanophore stripes along the
dorsal and ventral edges of the myotomes, and along the horizontal myoseptum
(Milos and Dingle, 1978a;
Kelsh et al., 2000). The early
larval pigment pattern remains essentially unchanged for about two weeks,
until the onset of pigment pattern metamorphosis. At this time, new
melanophores appear over the flank in regions not previously occupied by these
cells, and during the following two weeks, the pigment pattern is transformed
into that of the adult (Fig. 1)
(Kirschbaum, 1975;
Johnson et al., 1995;
Parichy et al., 2000b).
Genetic and cellular analyses demonstrate that new melanophores arising at
metamorphosis differentiate from latent precursors or stem cells of
presumptive neural crest origin (Johnson
et al., 1995; Parichy and
Turner, 2003b); such melanophores also play a crucial role in
pigment pattern regeneration (Goodrich and
Nichols, 1931; Rawls and
Johnson, 2000; Rawls and
Johnson, 2001). Although previous studies provide compelling
evidence that metamorphic and regenerative melanophores are derived from
post-embryonic latent precursors, specific markers for these cells have not
been demonstrated, and their locations, potencies and developmental
requirements remain largely unknown. Given these caveats, we refer to these
post-embryonic melanophores simply as `metamorphic' melanophores.
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;
Quigley and Parichy, 2002;
Parichy, 2003). Besides
horizontal stripes of varying width and number, these species exhibit vertical
bars and uniform pigment patterns in which melanophores are dispersed evenly
over the flank (Fig. 1). This
interspecific variation provides an opportunity to dissect the genetic and
cellular mechanisms underlying naturally occurring phenotypic variation in an
adult trait of neural crest origin. In this study, we ask whether adult melanophore stripes develop similarly across species, and in particular, whether the relative roles of neural crest-derived early larval melanophores and metamorphic melanophores have been maintained during evolution. To address this question, we first examine D. nigrofasciatus (Fig. 1), a species having fewer melanophores and stripes than D. rerio, but in which stripes that do develop are similar to those of D. rerio. Whereas stripes in D. rerio arise almost entirely from metamorphic melanophores, we show that stripes in D. nigrofasciatus arise from fewer metamorphic melanophores and an increased number of neural crest-derived early larval melanophores that persist into the adult. This interspecific variation led us to test the relative roles of these melanophore lineages during pigment pattern development in several additional species. These analyses demonstrate that a primary role for metamorphic melanophores in adult pigment pattern formation is likely to be ancestral for Danio, and that D. nigrofasciatus exhibits a unique, derived reduction in these cells, with a corresponding increased contribution of early larval melanophores to the adult pigment pattern. We further demonstrate that evolutionary changes within D. nigrofasciatus are extrinsic (non-autonomous) to the melanophore lineages, and we identify a candidate genetic pathway for mediating this change. These analyses highlight the potential for studies of D. rerio and its relatives to reveal basic mechanisms of post-embryonic neural crest development.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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), and may
represent a variant or subspecies; for ease of presentation we refer to these
fish simply as `D. kyathit' in the text, but to acknowledge the
uncertainty of their precise taxonomic affinity, we refer to the fish as
`D. aff. kyathit' in the phylogram. D. rerio
mutants have already been described: albinob4
(Kelsh et al., 2000),
sox10 (colourless)
(Dutton et al., 2001),
endothelin receptor b1 (ednrb1, roseb140)
(Parichy et al., 2000a),
tfap2a (lockjawts213)
(Knight et al., 2003;
Knight et al., 2004),
mitfa (nacrew2)
(Lister et al., 1999), and
pumaj115e1 (Parichy
and Turner, 2003b; Parichy et
al., 2003). Additional D. rerio mutants were derived from
on-going mutagenesis screens (D.M.P., E.H.B. and E.L.M., unpublished).
Interspecific complementation tests were performed as previously described
(Parichy and Johnson, 2001) by
in vitro fertilization. Because of difficulties obtaining fertilizable eggs
from heterospecific danios, most complementation tests were performed using
D. rerio females and heterospecific males. When the identities or map
positions of D. rerio mutant loci were known, heterozygotes were used
for generating interspecific hybrids, to randomize effects across unlinked
loci, and progeny were genotyped for the presence or absence of the mutant
allele by PCR (primers and diagnostic single nucleotide polymorphisms
available on request). Finally, puma mutant D. rerio are
temperature-sensitive, with growth rate-dependent pigment pattern defects at
25°C, moderate pigment pattern defects at 28.5°C, and more severe
defects at 33°C (Parichy et al.,
2003); tester puma hybrids were reared at the
intermediate temperature of 28.5°C to avoid mortality owing to stresses at
the higher temperatures.
Nomenclature for pigment pattern elements at larval and adult stages
Previous studies defined pigment pattern elements in D. rerio
(Parichy and Johnson, 2001;
Parichy and Turner, 2003b),
including: early larval dorsal, lateral and ventral melanophore
stripes (ELD, ELL, ELV); adult first-developing (primary) dorsal and
ventral melanophore stripes (1D, 1V); and later-developing (secondary) dorsal
and ventral melanophore stripes (2D, 2V). Additionally, between adult
melanophore stripes are xanthophore-rich `interstripe' regions. For
simplicity, we use the term `stripes' to refer exclusively to the adult
primary melanophore stripes (1D, 1V), unless indicated otherwise.
Microscopy, imaging and quantitative analyses
To examine melanophore behavior, we repeatedly imaged individual larvae
during pigment pattern metamorphosis, allowing us to follow the appearance,
disappearance and migration of individual melanophores
(Parichy et al., 2000b;
Parichy and Turner, 2003b).
Individually reared fish were anesthetized with MS222 (Sigma) and imaged every
24 hours using an Olympus SZX-12 stereozoom microscope. To ensure that we
could follow cells at the edges of the flank, all fish were imaged lying
parallel to the camera, and also on a specially constructed stand providing an
angle 30° from normal. Images were transferred to Adobe Photoshop CS for
analysis, in some cases in conjunction with the FoveaPro 3.0 image processing
and analysis package (Reindeer Graphics).
Individual melanophores were tracked as previously described
(Parichy et al., 2000b;
Parichy and Turner, 2003b),
with newly differentiated melanophores clearly distinguishable from
pre-existing melanophores by their initially lighter melanization (and in some
instances different color, see below). We identified individual melanophores
present in the early larval pigment patterns, then examined the fates of these
cells by examining their positions in sequential images. In following early
larval melanophores through metamorphosis, we could not formally observe cell
division in static image series, so we tracked only one presumptive daughter
following likely mitoses. Thus, our counts and estimated proportions of early
larval melanophore contributions to later stages in D. nigrofasciatus
are conservative and may underestimate true values to some degree. For
comparisons of early larval and metamorphic melanophore numbers between
species, we defined an area of interest bounded anteriorly by the anteriormost
anal fin ray insertion and posteriorly at two myotomes anterior to the caudal
peduncle. We counted individual early larval melanophores unilaterally within
this region. We determined total adult melanophore numbers within this region,
either within the adult ventral primary melanophore stripe, if present, or at
an equivalent dorsoventral position as observed in D. rerio, with a
height defined arbitrarily as one-quarter the flank height, as measured at the
anterior boundary. In final images from each individual, all melanophores in
the region of interest were marked and counted, either by eye or by the Count
plug-in of FoveaPro 3.0. Numbers of metamorphic melanophores were thus
calculated as the difference between the total numbers of melanophores
identified in the final images, and the numbers of melanophores that had been
followed into the region of interest from early larval stages. Statistical
analyses were performed with JMP 5.0.1a Statistical Software (SAS Institute,
Cary, NC). Additional information on quantitative image analyses is available
on request.
Cell transplantation and genetic mosaic analysis
We transplanted cells between mid-blastula stage [3.3-3.8 hours
post-fertilization (hpf)] D. rerio and D. nigrofasciatus
embryos, using a Narishige IM-9B micrometer-driven microinjection apparatus
mounted on a Narishige micromanipulator. We placed embryos in agar-lined
dishes containing 10% Hanks solution plus 1% penicillin/streptomycin, and
dechorionated embryos with fine forceps. We transplanted 20-100 cells into
each recipient and reared chimeric individuals through adult stages. To
identify donor D. rerio cells in D. nigrofasciatus hosts, we
used donors that were transgenic for EGFP driven by a ubiquitously expressed
D. rerio ß-actin promoter, kindly provided by Ken Poss
(Parichy and Turner, 2003a;
Parichy et al., 2003). To
identify donor D. nigrofasciatus melanophores in D. rerio
hosts, we used hosts mutant for albino or nacre
(mitfa), which fail to develop melanin and melanophores,
respectively. Both of these mutant loci normally act autonomously to the
melanophore lineage, as revealed previously
(Lin et al., 1992;
Lister et al., 1999;
Parichy and Turner, 2003a) and
confirmed in control experiments performed for the present analyses (data not
shown). Previous studies reveal minimal local correlation between the
distributions of pigment cells and other tissues in genetic mosaics examined
at metamorphic and adult stages
(Maderspacher and Nusslein-Volhard,
2003; Parichy and Turner,
2003a; Parichy et al.,
2003). We confirmed that donor melanophores typically develop
independently of other local donor tissues in a subset of chimeras in which
donor embryos were injected with rhodamine dextran prior to the four-cell
stage, then were examined for the distribution of melanophores and other
tissues at 4 dpf (data not shown). We sorted chimeras at 3 dpf for the
presence or absence of donor melanophores, and as larvae approached
metamorphosis, we repeatedly imaged individual larvae to follow the behavior
of early larval melanophores and to assess the distribution of metamorphic
melanophores. Survival rates for interspecific chimeras were typically 5-10%
of that observed for comparable experiments involving only D. rerio
(Parichy and Turner, 2003a;
Parichy et al., 2003),
suggesting some species incompatibilities; 
1% of chimeras were
informative for analyses of pigment pattern formation (see Results).
In situ hybridization and histology
We used in situ hybridization to detect transcripts for melanophore lineage
markers, as described previously (Parichy
et al., 2000a; Parichy et al.,
2000b; Parichy et al.,
2003). Larvae were fixed briefly in 4% paraformaldehyde, 1% DMSO
in PBS, decapitated, and then fixed overnight at 4°C. Larvae were
transferred to methanol, rehydrated to PBST (PBS with 0.2% Tween-20), then
treated for 20 minutes at room temperature with 20 µg/ml proteinase-K in
PBST containing 1% DMSO. Larvae were postfixed for 20 minutes at room
temperature in 4% paraformaldehyde, 0.005% glutaraldehyde, washed in PBST,
then washed three times in hybridization solution lacking tRNA and heparin.
Prehybridizations were performed overnight at 60°C in hybridization
solution (50% formamide, 5xSSC, 500 µg/ml yeast tRNA, 50 µg/ml
heparin, 0.2% Tween-20, 9.2 mM citric acid). Hybridizations were performed at
60°C over two nights, in fresh hybridization solution containing
digoxigenin-labeled riboprobes fractionated to 300 nucleotides. Larvae
were then washed twice, for 15 minutes each, in 2xSSCT, and three times,
for 2 hours each, in 0.2xSSCT at 60°C. After graded changes to PBST,
larvae were blocked overnight at 4°C in 2 mg/ml BSA, 5% heat-inactivated
calf serum in PBST, then incubated at 4°C over two nights in fresh
blocking reagent containing 1:5000 anti-digoxigenin alkaline
phosphatase-conjugated Fab fragments (Roche). Larvae were washed over two
nights in PBST, transferred to alkaline phosphatase buffer [100 mM Tris (pH
9.5), 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20], and the color
developed with NBT/BCIP.
To assay for tyrosinase activity, larvae were fixed for 2 hours in 4%
paraformaldehyde in PBS, rinsed three times in PBS, incubated in 0.1%
L-dopa (Sigma) for 1 hour to overnight, rinsed in PBS, then stored
in glycerol (Camp and Lardelli,
2001; McCauley et al.,
2004). We verified the specificity of the assay for melanoblasts
by the reduced staining on the flanks of metamorphosing nacre mutant
D. rerio, which have defects in the melanophore lineage
(Lister et al., 1999;
Parichy et al., 2000b), and we
verified that newly melanized (tyrosinase+) cells are not
macrophages by Neutral Red staining
(Herbomel et al., 1999) (data
not shown).
Phylogenetic analysis
We reconstructed phylogenetic relationships based on mitochondrial 12S and
16S rDNA sequences, obtained using standard methods and universal primers
(Kocher et al., 1989;
Palumbi et al., 1991).
12S: H1478, 5'-TGA CTG CAG AGG GTG ACG GGC GGT GTG T-3'; L1091, 5'-AAA AAG CTT CAA ACT GGG ATT AGA TAC CCC ACT AT-3'.
16S: 16Sar-L, 5'-CGC CTG TTT ATC AAA AAC AT-3'; 16Sbr-H, 5'-CCG GTC TGA ACT CAG ATC ACG T-3'.
Sequences were aligned using CLUSTAL-W, inspected by eye and edited as
necessary. We then analyzed combined 12S and 16S sequences (784 nucleotides)
using maximum likelihood estimation in PAUP* 4.0b10 for Macintosh
(Swofford, 2002). Maximum
likelihood analyses used a general time-reversible plus gamma model.
Substitution rate matrix, nucleotide frequencies, and among site rate
variation were estimated from the data by preliminary parsimony analyses using
a heuristic search strategy. Maximum likelihood, parsimony and distance
methods produced trees with the same topology. To estimate confidence values
for reconstructed nodes, we performed two independent analyses. First, we
performed 100 nonparametric bootstrap replicates using PAUP*.
Second, we performed a Bayesian analysis of the data using MrBayes
(Larget and Simon, 1999;
Huelsenbeck and Ronquist,
2001; Wilcox et al.,
2002), with 3000 replicate trees from 300,000 generations
following the approach to asymptotic likelihood values. Both approaches gave
nearly identical confidence values, which we report as percentages of
recovered trees in the phylogram (see Results).
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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),
for which stripes on the flank are superficially similar
(Fig. 1).
Closer inspection reveals about twice as many melanophores in D.
rerio than in D. nigrofasciatus
(Fig. 2A,C; see below).
Melanophore colors differ as well. In D. rerio, the dorsal and
ventral stripes consist almost entirely of grey-black melanophores. Yet,
occasional brownish melanophores occur at the ventral edge of the dorsal
stripe (Fig. 2A,B), where a few
melanophores derive not from latent precursors at metamorphosis, but from the
rearrangement of embryo-derived melanophores originally present in the early
larval lateral stripe along the horizontal myoseptum
(Parichy and Turner, 2003b).
In D. nigrofasciatus, however, both dorsal and ventral stripes
contain numerous brown melanophores (Fig.
2C,D), and melanophores are not present along the ventral myotome
edge (where the early larval ventral stripe had been). Melanophore color
variation is apparent transiently after metamorphosis, and is not equally
pronounced in all families; whether this variation reflects the age of the
melanin contained within the cells or some other biochemical difference is not
clear. Nevertheless, the differences in melanophore colors and their relative
frequencies in the adult pigment patterns of D. rerio and D.
nigrofasciatus led us to hypothesize that cryptic patterning variation
might underlie the superficially similar stripes between these species.
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;
Parichy and Turner, 2003b).
These analyses demonstrate that D. rerio stripes depend almost
entirely on melanophores that differentiate from latent precursors at
metamorphosis. By stark contrast, the superficially similar stripes of D.
nigrofasciatus arise by the reorganization of early larval melanophores,
with far fewer metamorphic melanophores.
In D. rerio, the onset of pigment pattern metamorphosis is marked
by the differentiation of single `pioneer' metamorphic melanophores over the
middle of most ventral myotomes (Fig.
3A). Subsequently, metamorphic melanophores differentiate widely
over the myotomes, between the early larval stripes
(Fig. 3B,C). The adult primary
stripes become increasingly apparent (Fig.
3D), as initially dispersed metamorphic melanophores migrate short
distances to the sites of stripe formation, and as additional metamorphic
melanophores differentiate within the stripes themselves
(Fig. 3C,D). A few early larval
melanophores migrate from the horizontal myoseptum to join the dorsal adult
primary melanophore stripe (Fig.
3D,E), but most remain in place and eventually are lost
(Parichy and Turner, 2003b).
As fish approach the end of metamorphosis, a juvenile pattern emerges, with
adult dorsal and ventral primary melanophore stripes consisting almost
entirely of melanophores that have differentiated from latent precursors
during metamorphosis (Fig.
3E,E').
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Ancestral role for metamorphic melanophores in adult pigment pattern development and derived patterning mechanisms in D. nigrofasciatus
The relative contributions to adult pigment patterns of early larval
melanophores and metamorphic melanophores could vary continuously across
species. Alternatively, either the D. rerio or the D.
nigrofasciatus mode could be typical. To distinguish between these
possibilities, and to determine which, if either, mode is ancestral and which
is derived, we sought to examine pigment pattern metamorphosis in additional
species.
Because danio relationships remain poorly understood, we first sequenced
12S and 16S rDNA from additional taxa to infer phylogenetic relationships
(Fig. 5). These analyses
confirm the close relationship between D. rerio and D.
nigrofasciatus, as well as D. kyathit
(Fig. 1). The phylogeny also
supports a split between Danio and Devario [formerly within
Danio (Fang, 2003)].
Moreover, these data reveal additional pigment pattern diversity within
Danio (as defined in Fig.
5): these fish have been known to have horizontal stripes, spots,
uniform patterns, and more complex pigment patterns; Danio choprae
adds vertical barring to the repertoire
(Fig. 1).
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D. rerio mutants identify a candidate pathway for metamorphic melanophore reduction and early larval melanophore morphogenesis in D. nigrofasciatus
D. rerio mutants can identify genes and pathways that contribute
to interspecific pigment pattern differences
(Parichy and Johnson, 2001).
Given the reduced number of metamorphic melanophores in D.
nigrofasciatus compared to D. rerio, we investigated whether
genes isolated as D. rerio mutants with defects in melanophore
development also contribute to the difference between species. We used
interspecific hybrids to test for complementation of D. rerio mutant
alleles by crossing mutant D. rerio to D. nigrofasciatus and
comparing these tester (mutant) hybrids to control (wild-type) hybrids. Tester
hybrids exhibiting fewer melanophores than controls identify genes that may
contribute to the interspecific difference, whereas tester hybrids that have
similar melanophore numbers to controls identify genes less likely to have
major effect roles.
Control hybrids between wild-type D. rerio and D.
nigrofasciatus have phenotypes intermediate between species. Whereas
melanophore numbers in primary adult stripes are increased over D.
nigrofasciatus and are closer to D. rerio, melanophore numbers
in secondary adult stripes, and the total numbers of stripes, are closer to
D. nigrofasciatus than D. rerio
(Fig. 8A)
(Parichy and Johnson, 2001).
Comparing adult hybrid phenotypes does not reveal gross non-complementation of
the recessive melanophore mutants sox10ut.r13e1,
tfap2ats213, bonaparteut.r16e1,
cezanneut.r17e1, degasut.r18e1,
oberonj198e1, pissarrout.r8e1,
picassout.r2e1, primrosej199, pumaj115e1
or seuratut.r15e1 (e.g.
Fig. 8), adding to the
previously excluded loci ednrb1, fms, kit, mitfa, leopard, fritz and
jaguar (Parichy and Johnson,
2001). Thus, genes contributing to the differences in the final
numbers of adult melanophores between species either are not likely to be
represented in this collection of 18 D. rerio pigment pattern
mutants, or differences in allelic strengths are not sufficient to reveal
non-complementation.
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We first investigated whether any of several D. rerio mutants
exhibiting stripes dorsally and spots ventrally, as in D.
nigrofasciatus, might have similar modes of pigment pattern metamorphosis
to D. nigrofasciatus. Examination of one of these mutants,
ednrb1 (Parichy et al.,
2000a), revealed little contribution of early larval melanophores
to the adult pigment pattern, unlike in D. nigrofasciatus
(Fig. 9A-D, and data not
shown). Thus, a similarity of pigment pattern elements does not predict the
underlying mode of pigment pattern metamorphosis.
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). To see whether puma or picasso
contribute to the difference between species, we tested whether early larval
melanophore fates and adult stripe compositions in tester hybrids differ from
those of control hybrids. Repeated imaging of hybrid larvae reveals that early
larval melanophores make little contribution to the adult stripes in control
(wild-type) hybrids (Fig. 9M-P;
n=9), tester picasso hybrids, or tester hybrids for two
additional mutants, pissarro and seurat (n=4,11 and
6, respectively; data not shown). In tester puma hybrids
(n=9), however, a greater number of early larval melanophores leave
their initial positions along the myotome edges and relocalize over the flank
(Fig. 9Q-T), similar to
puma mutants and D. nigrofasciatus
(Fig. 9E-L,
Fig. 3F-J). These hybrids have
somewhat reduced numbers of metamorphic melanophores initially, but this
deficit is regulated by adult stages so that adults have total melanophore
complements that are indistinguishable from control hybrids. The developmental
non-complementation phenotype of tester puma hybrids identifies
puma or genes in its pathway as candidates for contributing to the
evolutionary reduction of metamorphic melanophores, and increased contribution
of early larval melanophores to the D. nigrofasciatus adult pigment
pattern. These results indicate that differences in total numbers of adult melanophores between D. rerio and D. nigrofasciatus are not likely to be explained by differences at loci already isolated as D. rerio melanophore mutants. Moreover, similarity of adult pigment pattern alone is not a good predictor for the underlying mode of pigment pattern metamorphosis. By contrast, interspecific complementation tests for melanophore morphogenesis suggest a role for puma or its pathway in determining the relative contributions of metamorphic melanophores and neural crest-derived early larval melanophores to the adult pigment patterns of D. rerio and D. nigrofasciatus.
Reduction of metamorphic melanophore lineage in D. nigrofasciatus
The reduction in metamorphic melanophores in D. nigrofasciatus
could reflect a failure to recruit committed melanophore precursors
(melanoblasts) from uncommitted latent precursors or stem cells during
metamorphosis. For example, puma mutant D. rerio exhibit
severe reductions in metamorphic melanoblasts compared with wild-type D.
rerio (Parichy et al.,
2003). If the same pathway affected in puma mutant D.
rerio has evolved between D. rerio and D.
nigrofasciatus, then fewer melanoblasts should be observed in D.
nigrofasciatus compared with wild-type D. rerio. Alternatively,
fewer metamorphic melanophores in D. nigrofasciatus could reflect a
later block in this lineage, with similar numbers of melanoblasts being
recruited from latent precursors then failing to terminally differentiate as
melanophores. To distinguish between these possibilities, we used molecular
markers and histological assays to compare D. rerio and D.
nigrofasciatus during metamorphosis.
Our examination of the melanophore lineage during metamorphosis reveals a
severe reduction in the number of melanoblasts in D. nigrofasciatus,
suggesting an early block in metamorphic melanophore development. We examined
the distribution of cells expressing transcripts for two molecular markers,
dopachrome tautomerase (dct) and tyrosinase
(tyr), which encode enzymes required for melanin synthesis and thus
identify committed melanophore precursors (as distinct from latent stem cells)
(Kelsh et al., 2000;
Camp and Lardelli, 2001). We
observed fewer dct+ and tyr+ cells
throughout metamorphosis in D. nigrofasciatus compared to D.
rerio (Fig. 10).
Importantly, however, we observed strong staining for each marker in fully
differentiated melanophores, and in the more rare, unmelanized cells, in
D. nigrofasciatus, demonstrating the efficacy of these probes in this
cross-species comparison.
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).
Comparing the same regions of the flank before and after L-dopa incubation, we
observe newly melanized cells in D. rerio larvae, but only 10%
as many of these cells in D. nigrofasciatus larvae
(Fig. 11). These findings
support the conclusion that D. nigrofasciatus exhibit fewer
melanoblasts during metamorphosis than D. rerio do.
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Differences between D. rerio and D. nigrofasciatus are non-autonomous to melanophore lineages
The different modes of pigment pattern metamorphosis in D. rerio
and D. nigrofasciatus could reflect evolutionary changes that are
intrinsic (autonomous) or extrinsic (non-autonomous) to melanophore lineages.
Although species differences have been attributed to intrinsic factors
(Twitty and Bodenstein, 1939;
Rawles, 1948;
Schneider and Helms, 2003),
the extensive migrations and cellular interactions during neural crest and
melanophore development imply many opportunities for extrinsic factors to
generate differences in form as well
(Erickson and Perris, 1993;
Parichy, 1996;
Halloran and Berndt, 2003). To
distinguish between these possibilities, we examined melanophore behaviors and
patterns in genetic mosaics. These analyses demonstrate a primary role for
extrinsic factors in determining early larval melanophore contributions to
adult stripes, as well as the positions of adult stripes on the flank.
We transplanted cells from D. nigrofasciatus to D. rerio,
and then reared chimeras through metamorphosis
(Parichy and Turner, 2003a;
Parichy et al., 2003). To
identify donor D. nigrofasciatus melanophores, we used D.
rerio hosts mutant for the albino locus, which acts autonomously
to the melanophore lineage to promote melanization, but does not otherwise
affect melanophore development or pigment pattern formation
(Lin et al., 1992); D.
nigrofasciatus melanophores thus appear as the only melanized cells in a
field of unmelanized but otherwise normal melanophores
(Lin et al., 1992;
Parichy et al., 1999;
Kelsh et al., 2000). To assess
the mode of pigment pattern metamorphosis, we identified chimeras that
developed D. nigrofasciatus early larval melanophores, then we
repeatedly imaged these individuals through metamorphosis.
We predicted that if species differences are autonomous to the melanophore
lineages, then donor D. nigrofasciatus early larval melanophores
should contribute to the adult ventral melanophore stripe (as in D.
nigrofasciatus); if differences between species are non-autonomous to the
melanophore lineages, then donor D. nigrofasciatus early larval
melanophores should fail to contribute to this stripe (as in D.
rerio). Fig. 12A-D shows
a representative D. nigrofasciatus
D. rerio chimera.
Donor D. nigrofasciatus early larval melanophores are present within
the early larval stripe along the ventral myotomes but do not contribute to
the adult ventral stripe. Thus, early larval melanophore morphogenesis
resembles that of D. rerio rather than that of D.
nigrofasciatus (compare with Fig.
3). Moreover, D. nigrofasciatus melanophores that
differentiated during metamorphosis did so at the normal location of D.
rerio stripes, rather than further ventrally as in D.
nigrofasciatus (compare with Fig.
3). These findings indicate that factors non-autonomous to the
melanophore lineages determine species differences in early larval melanophore
contributions to adult stripes, as well as the positions of adult stripes.
These results also obviate the identification of other donor D.
nigrofasciatus cells in D. rerio hosts, as the final
distributions of donor melanophores cannot easily be explained by a simple
coincidence of D. nigrofasciatus melanophores and other D.
nigrofasciatus donor tissues (which might have explained the alternative
result, had donor melanophores behaved like their own, donor species, rather
than the host species).
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D. nigrofasciatus chimeras
(n>40), we never observed donor D. rerio melanophores in
D. nigrofasciatus hosts, although we frequently observed
other donor tissues (e.g. myotomes, nerves, skin; data not shown). Similar
studies in D. rerio yielded comparable frequencies of these tissues,
but also melanophores (Parichy and Turner,
2003a; Parichy et al.,
2003). These data suggest that D. nigrofasciatus hosts
may be compromised in their ability to support D. rerio donor
melanophores, supporting a model in which species differences in metamorphic
melanophore differentiation are non-autonomous to this lineage as well.
Finally, we investigated whether evolutionary changes in interactions
between melanophores themselves might contribute to the different metamorphic
modes between species. We reasoned that a reduction in the numbers of
metamorphic melanophores, and thus reduced contact inhibition of movement
(Tucker and Erickson, 1986),
might allow early larval melanophores to leave their initial positions during
metamorphosis in D. nigrofasciatus. To test this possibility, we
transplanted D. nigrofasciatus cells to nacre mutant D.
rerio hosts. nacre mutants lack melanophores owing to an
inactivating mutation in mitfa, which normally acts autonomously to
the melanophore lineage (Lister et al.,
1999). We predicted that if changes in melanophore-melanophore
interactions alone are responsible for species differences, then D.
nigrofasciatus early larval melanophores in nacre mutant hosts
should contribute to the adult ventral stripe (as in D.
nigrofasciatus). If other factors contribute to the species differences,
the D. nigrofasciatus early larval melanophores should fail to
contribute to this stripe (as in D. rerio).
Fig. 12F-I shows a D.
nigrofasciatusnacre mutant D. rerio chimera.
Repeated imaging demonstrates that donor D. nigrofasciatus early
larval melanophores do not contribute to the adult ventral stripe, which forms
at a position similar to that seen in D. rerio. These data
demonstrate that factors extrinsic to melanophore lineages contribute to
differences in pigment pattern metamorphosis between D. rerio and
D. nigrofasciatus.
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Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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